High dose methotrexate with leucovorin rescue is currently being used in a number of clinical protocols for the treatment of metastatic osteogenic sarcoma and a variety of other neoplasms. In both L1210 leukemia and sarcoma 180, Sirotnick and coworkers have clearly demonstrated that high dose methotrexate and leucovorin rescue can be therapeutically optimized by delaying leucovorin administration and minimizing leucovorin doses. The schemes for leucovorin rescue presently being used clinically have been developed on an empiric basis. A thorough knowledge of the pharmacology and pharmacokinetics of leucovorin would enable clinicians to develop a more rational approach to limiting the doses of leucovorin and improving the therapeutic index of methotrexate/leucovorin therapy. In order to accomplish this, an accurate, sensitive assay for leucovorin and 5-methyl THF (the active metabolite) have been developed in plasma. Using this assay, the disposition and elimination of leucovorin and 5-methyl THF will be studied in humans. In particular, emphasis will be placed on studying the plasma protein binding of leucovorin and drug-drug interactions between leucovorin and methotrexate, antibiotics, antiemetics and other anti-neoplastic agents. The results of this study will be used to develop a more rational approach towards the use of leucovorin in an effort to minimize leucovorin doses and to optimize the therapeutic efficacy of high dose methotrexate with leucovorin rescue therapy.